Takeshi Senga

Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration.

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell–cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.

PCNA Is a Cofactor for Cdt1 Degradation by CUL4/DDB1-mediated N-terminal Ubiquitination *

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Nitric Oxide Controls Src Kinase Activity through a Sulfhydryl Group Modification-mediated Tyr-527-independent and Tyr-416-linked Mechanism

c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator,S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N2O3 scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitro labeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivo labeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na3VO4 did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N2O3-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N2O3-provokedS-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.

YAP is essential for tissue tension to ensure vertebrate 3D body shape

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D’Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

Secretion of matrix metalloproteinase‐9 by the proinflammatory cytokine, IL‐1β: a role for the dual signalling pathways, Akt and Erk

Background: Matrix metalloproteinases including MMP‐9 mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP‐9 up‐regulation by inflammatory cytokines involve interactions between several transcription factors including activator protein‐1 and NFκB. The upstream regulatory pathways are less well understood.

Nek2 as a novel molecular target for the treatment of breast carcinoma

We investigated the role of Nek2, a member of the serine‐threonine kinase family, in the tumorigenic growth of breast carcinoma. Increased expression of Nek2 was observed in all breast carcinoma cell lines examined (BT20, BT474, Hs578T, MCF7, MDA‐MB‐231, T47D, and ZR‐75‐1) by immunoblotting. By treatment with Nek2 short interfering RNA (siRNA), expression of Nek2 was clearly decreased in both estrogen receptor (ER)‐positive (MCF7) and ER‐negative (MDA‐MB‐231) breast carcinoma cell lines. Cell growth, colony formation in soft agar, and in vitro invasiveness of these cell lines were substantially suppressed by Nek2 siRNA treatment. In a xenograft nude mouse model with subcutaneous implantation of MCF7 or MDA‐MB‐231, subcutaneous injection of Nek2 siRNA around the tumor nodules resulted in a reduction of tumor size compared with those of control siRNA injection. Taken together, Nek2 appears to play a pivotal role in tumorigenic growth of breast carcinoma cells, and could be a useful therapeutic molecular target for the treatment of breast carcinoma both in ER‐positive and ER‐negative cases. (Cancer Sci 2009; 100: 111–116)

S-Nitrosylation at Cysteine 498 of c-Src Tyrosine Kinase Regulates Nitric Oxide-mediated Cell Invasion

Nitric oxide (NO) plays a pivotal role in tumorigenesis, particularly with relation to cancer cell invasion and metastasis. NO can reversibly couple to cysteine thiols to form an S-nitrosothiol, which regulates the enzymatic activities of target proteins. c-Src is a tyrosine kinase that promotes cancer cell invasion and metastasis. Interestingly, c-Src can be activated by NO stimulation. However, mechanisms by which NO stimulates Src kinase activity have not been elucidated. We report here that NO causes S-nitrosylation of c-Src at cysteine 498 (Cys498) to stimulate its kinase activity. Cys498 is conserved among Src family kinases, and Cys506 of c-Yes, which corresponds to Cys498 of c-Src, was also important for the NO-mediated activation of c-Yes. Estrogens may work synergistically with NO to induce the proliferation and migration of many kinds of breast cancer cells. For example, β-estradiol induces the expression of endothelial nitric synthase and production of NO in MCF7 cells. We found that activation of c-Src in MCF7 cells by β-estradiol stimulation was mediated by the S-nitrosylation of Cys498. In addition, we report that disruption of E-cadherin junctions and enhancement of cell invasion by β-estradiol stimulation was mediated by NO-dependent activation of c-Src. These results identify a novel signaling pathway that links NO and Src family kinases to cancer cell invasion and metastasis.

A Role for Focal Adhesion Kinase Signaling in Tumor Necrosis Factor-α–Dependent Matrix Metalloproteinase-9 Production in a Cholangiocarcinoma Cell Line, CCKS1

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) stimulation of CCKS1, a cell line established from cholangiocarcinoma with i.p. dissemination, dramatically increased matrix metalloproteinase-9 (MMP-9) production and tumor invasion. We investigated the role of focal adhesion kinase (FAK) in TNF-alpha-dependent production of MMP-9 in CCKS1 and FAK-null mouse fibroblast cells. TNF-alpha stimulation of CCKS1 or wild-type fibroblasts substantially activated FAK phosphorylation and increased MMP-9 production. In contrast, FAK-null fibroblasts could not respond well to TNF-alpha stimulation. Conditional expression of wild-type FAK in FAK-null cells restored the TNF-alpha-dependent production of MMP-9. TNF-alpha treatment activated the kinase activity of FAK and its phosphorylation especially at Y397 and Y925. Phosphorylated FAK accumulated at focal adhesions and formed a complex with growth factor receptor binding protein 2 and SOS. In contrast, Y397F FAK and Y925F FAK, whose Y397 and Y925 were replaced with phenylalanine, respectively, as well as KD FAK, whose kinase was inactivated, could not restore the MMP-9 production. In addition, small interfering RNA against FAK drastically suppressed the TNF-alpha-dependent production of MMP-9 and inhibited the TNF-alpha-dependent invasion of CCKS1. Taken together, our results suggest the pivotal role of FAK in TNF-alpha-dependent production of MMP-9 and subsequent activation of tumor invasion.

FAK Signaling in Neoplastic Disorders

Abstract:  Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, is frequently overexpressed in various tumors and its expression shows good correlation with the progression of tumor. FAK is involved in a diverse range of critical cellular events including spreading, proliferation, migration, and invasion. In addition to these cellular functions, we found that FAK signaling played a critical role in the production of matrix metalloproteinases (MMP) such as MMP‐2 and MMP‐9 and subsequently activated tumor invasion. Moreover, we found that tumor necrosis factor‐α (TNF‐α), a proinflammatory cytokine that acts as an endogenous tumor promoter, activated FAK signaling and enhanced tumor invasion. Since the tumor microenvironment that is largely orchestrated by cytokines is a critical component of tumor progression, these results suggest the importance of FAK as a signaling molecule involved in tumorigenesis. Here, we review the general structure and binding partners of FAK, its regulatory mechanism, and expression in tumors. By summarizing our recent studies, we focus on the critical role of FAK that links cancer with inflammation.

Three-dimensional cell culture array using magnetic force-based cell patterning for analysis of invasive capacity of BALB/3T3/v-src

A three-dimensional (3D) cell culture system has been fabricated using a magnetic force based cell patterning method, demonstrating a facile approach for the analysis of invasive capacity of BALB/3T3/v-src using an magnetic force and magnetite nanoparticles. The 3D cell patterning was performed using an external magnetic force and a pin holder, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns, resulting in the development of a 3D in vitro culture model. The cells embedded in type I collagen showed a compacted, spheroid like configuration at each spot, and distinct, accelerated cell growth was observed in cancer model cells compared with the control cells. The developed 3D cell culture array was applied to the susceptibility assay of the GM6001 matrix metalloproteinase (MMP) inhibitor, a collagenase inhibitor; a distinct suppression of cell proliferation was observed, while little change was observed in 2D. The developed 3D cell culture array system is useful to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion.